Infectious diseases have re-emerged as a major health problem in North America, in part due to the widespread emergence of antibiotics resistance. The mechanisms of defense against intracellular parasites, and the bacterial strategies underlying survival and replication in host phagocytes remain poorly understood. A better understanding of host defenses against such infections may suggest new strategies for intervention in these diseases. Using a genetic approach, we have identified a new component (Nramp1) of antimicrobial defenses of phagocytes. Mutations at Nramp1 in mice cause susceptibility to several intracellular infections, and polymorphic variants at human NRAMP1 are also associated with susceptibility to Mycobacterial infections in areas of endemic disease. Nramp1 is expressed at the membrane of bacterial phagosomes formed in macrophages and neutrophils, where it functions as an efflux pump to remove from the phagosomal space divalent metals essential for microbial replication. The current proposal describes a series of experiments aimed at better understanding the mechanism of Nramp1 transport, including the identification of the preferred substrate at the phagosomal membrane. We also wish to identify the metal dependent biochemical pathways and proteins in Mycobacterium species that are essential for survival in macrophages and that are inhibited by Nramp1 action. Finally, we have recently mapped 2 new loci (Trl-3, Trl-4) that control, in an Nramp1-independent fashion, the pulmonary replication of Mycobacterium tuberculosis in mice. We propose to characterize in congenic mouse lines the physiological responses, cell populations, and biochemical pathways of the host that are regulated by Trl-3 and Trl-4. The positional cloning of the underlying genes is also proposed. These combined studies may suggest new avenues and provide novel targets for intervention in infectious diseases.